Phosphorylation of serine, threonine, and tyrosine residues is a major mechanism for regulating protein function in eukaryotic cells. Protein kinases, the enzymes that catalyze these reactions, regulate all aspects of cell physiology and have thus emerged as therapeutic targets for a variety of human diseases. Small-molecule inhibitors of the Abelson tyrosine kinase (Abl) and the epidermal growth factor receptor (EGFR), for example, have been developed into clinically useful anti-cancer drugs. In addition to providing potential drug leads, selective inhibitors promise to increase the understanding of the cellular roles of protein kinases, most of which are poorly understood. Nearly all kinase inhibitors target the highly conserved adenosine triphosphate (ATP) binding site. Because the ATP binding sites are structurally similar even in divergent kinase domains, the rational design of inhibitors that selectively target even a subset of the ˜500 human kinases is a daunting challenge.
One attribute that makes protein kinases attractive drug targets is their ATP-binding site, a deep, hydrophobic cleft at the interface of two conserved subdomains. Many small molecules have been discovered that bind to this site with high affinity. However, because the ATP-binding sites of all protein kinases are highly similar, it has been difficult to design selective inhibitors that specifically target one or a few of the 500 human protein kinases.
The Rsk serine/threonine protein kinases have critical functions in the Ras/MAP kinase signaling pathway, a pathway which is deregulated in many human cancers. Of the four Rsk isoforms (Rsk1-4), Rsk1 and Rsk2 are the best characterized. Rsk1 and Rsk2 are directly activated by the MAP kinases, ERK1 and ERK2. Known substrates of Rsk1,2 include transcription factors involved in cell growth and differentiation (e.g. CREB, c-fos, estrogen receptor) and apoptosis (NF-κB). Rsk1,2 have thus been implicated in transcriptional control downstream of Ras and ERK1,2.
Rsk1-4 are unusual protein kinases in that they have two kinase domains, the NTD (NH2-terminal domain) and the CTD (CO2H-terminal domain). All Rsk substrates that have been characterized thus far are phosphorylated by the NTD. Downstream signaling by the NTD requires at least three sequential phosphorylation events: (1) phosphorylation of the CTD activation loop (T573) by ERK1,2; (2) intramolecular phosphorylation of a linker region (S380) by the CTD, which creates a docking site for the kinase PDK1; (3) phosphorylation of the NTD activation loop (S221) by PDK1.